CN110382713A - Improved epigenetic immunocytochemistry method - Google Patents
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Abstract
The present invention relates to improved epigenetic blood and immunocytochemistry method and respective application and kits.
Description
The present invention relates to improved epigenetic blood and immunocytochemistry method and respective application and kits.
Background of invention
The physiological equilibrium for deviateing cell immune system indicates various diseases, and therefore constitutes the weight of diagnosis and patient-monitoring
It measures.Flow cytometry (FCM) is the best approach accordingly measured at present, is provided about cell class to be determined
The accuracy of type and flexibility (1).However, although hematology analyzer used in diagnostic test room is more advanced and logistics environment
Process needed for adapting to extensively, but the applicability of this method is limited.
Cell count based on FCM needs to have the fresh of complete leucocyte, anticoagulation or saves good blood sample.
Even if still suggesting rapid operation using fresh sample, because analysis time will affect after blood extracts, initially a few houres start
Cell deterioration result.Further, since biology, technology and the variation of operation, the standardization of FCM is still one and great chooses
It fights (2,3,4,5), and not yet realizes standardized measurement (6,7) completely.However, the challenge of most critical is, and not all medical treatment
It can get fresh using all guarantees or save good blood sample, flow cytometry cannot be applied in these cases.
For example, determining the CD4 depending on patient to the treatment of HIV infection patient+T helper cell counts.It is being lower than 500
T helper cell/microlitre frequency under, it is strong to suggest carrying out antiretroviral treatment, and lower than 200 cells/microlitre
Level under be necessary.In resource-starved country, it cannot be frequently carried out continuously the rural area that blood is extracted and measured, fitted
When diagnostic assessment be frequently subjected to hinder.Therefore, it is usually based only upon the relevant clinical symptoms of HIV to start to treat, this may cause
Undesirable result (8,9).
Another example is neonatal screening.Collect the Guthrie card that punctures from heel, and be used to detect it is serious,
Recoverable birth defects.These cards cannot be used for flow cytometry, opposite T cell receptor resecting loop (TREC) by with
It is screened in PID.TREC analysis preferentially detects new thymus gland and moves out cell (main T cell hypotype in newborn's peripheral blood).However,
The technology is only limitted to T- (KREC) cell and nearest B- (KREC) cell, but is not suitable for the identification of such as CD4 or CD8 subgroup
Analysis, and other cell types can not be detected, such as NK cell.Therefore, the TREC analysis in newborn's analysis is only used for just
Walk screening.Antidiastole and patient-monitoring before and after therapeutic hematopoietic stem cell transplantation need to change technology and lead to
Overflow-type cell art carries out.
In order to overcome diagnosis limitation and the conversion of relevant technology, improved immune state appraisal procedure will be valuable.
It should steadily provide opposite and absolute cell count, also allow for using fresh, freezing or paper speck point (paper-
Spotted) blood.For the cell of each analysis, signal should be digitized, that is, each cell indicate a positive value or
Negative value, without arbitrarily defining " positive " threshold value.Such new method equally should be with automation, independently of operator
Mode carries out, and is less dependent on the variation of used reagent such as antibody.
In the first aspect of the invention, the above target is obtained by the quantitative methylation assay method of blood immune cells
It solves comprising following steps:
A) the human blood sample of the amount of determination is provided, it includes the diploid gene group DNA to quantitative blood immune cells；
B) recombinant nucleic acid for the bisulfite conversion simulated through computer is provided, it includes at least one demethylation marks
The sequence (" reference substance that it is GpC that all CpG dinucleotides of quasi- gene and at least one demethylation standard gene, which are inverted,
I")；
C) recombinant nucleic acid is provided, it includes at least one demethylation standard gene described in b) and b) described at least one
All CpG dinucleotides of a demethylation standard gene are inverted the genome sequence (" school of the demethylation for the sequence for being GpC
Quasi- object I ")；
D) recombinant nucleic acid is provided, it includes all CpG dinucleotides of at least one demethylation standard gene described in b)
The sequence (" calibration object I ") that it is GpC that acid, which is inverted,；
E) d of amount is determined to the sample addition described in a)) described in recombinant nucleic acid (" mark-on ")；
F) to the diploid gene group DNA of quantitative cell and c) and d) institute described in bisulfite processing a) is utilized
The recombinant nucleic acid stated, by unmethylated Cytosines at uracil；
G) using primer pair amplifies a) appropriate, b), c) and f) described in nucleic acid molecules, to generate amplicon；And
H) blood immune cells (BIC) of each volume sample are determined based on the analysis amplicon.
In the second aspect of the invention, the above target is solved by the quantitative methylation assay method of blood cell
Certainly comprising following steps:
A) human blood sample is provided, it includes the diploid gene group DNA to quantitative blood cell；
B) recombinant nucleic acid for the bisulfite conversion simulated through computer is provided, it includes at least one demethylation marks
Quasi- gene and at least one blood cell specific gene (" reference substance II ")；
C) recombinant nucleic acid is provided, it includes at least one demethylation standard gene described in b) and b) described at least one
The genome sequence (" caliberator II ") of the demethylation of a blood cell specific gene；
D) using bisulfite processing a) described in quantitative cell diploid gene group DNA and c) described in weight
Group nucleic acid, by unmethylated Cytosines at uracil；
E) using primer pair amplifies a) appropriate, b), c) and d) described in nucleic acid molecules, to generate amplicon；And
F) the demethylation percentage (DDC) of every kind of all cells is determined based on the analysis amplicon.
In terms of specific preferred third of the invention, the above target passes through for determining the methyl of immune cell type
The method for changing the absolute copy number of gene is resolved comprising:
A) implement the method for first aspect present invention as described above,
B) implement the method for the second aspect present invention as described above, and
C) identified BIC is multiplied with identified DDC.
The step of can implementing the method for the present invention in parallel or combined reaction bottle, as shown in Figure 2.
The present invention relates to the accurate quantifications of methylation data.This is related to all multicomponents and consideration:
1. internal standard object, such as the plasmid through computer simulation conversion.
2. the GAPDH standardizing agents (for example) with the methylated variant of specific gene in contrast
(normaliser)。
3. therefore, quantified according to using 1, by the GAPDH and specificity that force demethylation (but with identical copy
Number exists) gene of demethylation is compared the copy of all demethylations.
4. however, the above permission is really " absolute " quantitative, because the reference substance through computer simulation conversion is not
Corresponding biological sample (it is only converted in reaction bottle).
5. solving the problems, such as 4.It is referred to as GNoMs (the genome standardizing agents of methylation) based on addition and measurement,
All original series equimolars are included in plasmid by this, then carry out disposed of in its entirety (bisulfite processing and purifying).By
Exist in them with 1:1, therefore standard of perfection object, display can be simulated through computer after using the reference substance in 1 quantitative
The difference between methylation in situ.Using the factor, the methylation value of measurement can be corrected, this significantly improves results.
6. in addition, can be calculated using having the quantitative nucleic acid (plasmid) really of the standard gene containing inverted CG base
Any material damage in processing, this can further ameliorative way.
In principle, quantitative real-time PCR (qPCR) is such method, the apparent something lost based on high cell type specificity
Characterization is completed when passing (i.e. DNA methylation) marker (14,15,16).Technically, when using bisulfite processing DNA, not
The CpG dinucleotides of methylation is converted into TpGs (" TpG variant "), and the CpG- dinucleotides to methylate remains unchanged
(" CpG variant ").Therefore, epigenetic label is translated into the sequence for allowing discrimination and quantitative two kinds of variants by bisulfite conversion
Column information.QPCR measurement is insensitive to the loss of cell integrity in blood sample, because its DNA is highly stable entity.
Therefore, epigenetic cell quantification can be carried out to fresh peripheral blood, dry blood cake or any other sample, and it is saved
State does not have particular requirement.It assume that each site CpG that each cell is copied there are two, and in single given cell
Each site be entirely methylation or demethylation.In addition, being synthetically produced required component needed for qPCR, and compared to biological object
Relatively easy realize is standardized when the preparation of matter such as specific antibody.However, by today, such epigenetics method
Applicability has not proved out, it may be possible to due to lacking clearly defined cell type specificity biomarker and lacking implementation
The method of confirmation and absolute quantitation.The quantitative reference standard (17) for needing directly to represent bio-matrix of confirmation.Absolute quantitation is in base
Difficulty is caused in the technology of DNA, because the relationship between leucocyte DNA and blood volume is not fixed biologically, and
And the DNA recycling for carrying out autoblood extraction is sxemiquantitative.
Here, it is immune to inventor introduce one group of epigenetic quantitative for the major leukocyte group in human blood sample
The measuring method based on qPCR of cell-specific.The measurement is based on whole T cells, CD4+T cell, CD8+T cell, B-
The DNA methylation marker of cell and NK cell-specific.It is invaded in patient when there is fresh blood sample to can be used for such as HIV
CD4+When the measurement of T cell, the cell quantity of every microlitre of blood constitutes target criteria object.Therefore, it inventors herein proposes and is based on
Its cell type-specific epigenetic signal for the confirmation of immunocyte and the new system of absolute counting.Analyze survey
Determine the equivalence of concept and single biomarker and goldstandard FCM technology.However, when the volume of given blood does not determine accurately
When, such as the case where dried blood sample, relative measurement is more acurrate and should use.Therefore it in order to obtain extensive measurement range, invents
People tests various diagnostic applications herein, it is intended to determine absolute quantitation by analysis healthy donors and one group of HIV positive patient
Consistent degree, inventor also pass through identification one group of healthy newborn crowd in PID patient, determine the diagnosis from dry blood cake
Quality.
In preferred embodiment of the process according to the invention, method is integrated comprising:
A) the human blood sample comprising the diploid gene group DNA to quantitative blood cell of the amount of determination is provided；
B) recombinant nucleic acid that bisulfite simulate through computer converts is provided, it includes demethylation standard gene,
All CpG dinucleotides of the demethylation standard gene are inverted the sequence and blood cell specificity base for GpC
Cause；
C) recombinant nucleic acid is provided, it includes the genome sequence of the demethylation of the demethylation standard gene described in b),
Sequence that it is GpC that all CpG dinucleotides of the demethylation standard gene, which are inverted, and b) described in blood cell it is special
Property gene；
D) recombinant nucleic acid is provided, it includes all CpG dinucleotides of at least one demethylation standard gene described in b)
The sequence that it is GpC that acid, which is inverted,；
E) d of amount is determined to the sample addition described in a)) described in recombinant nucleic acid (" mark-on ")；
F) to the diploid gene group DNA of quantitative cell and c) and d) institute described in bisulfite processing a) is utilized
The recombinant nucleic acid stated, by unmethylated Cytosines at uracil；
G) using primer pair amplifies a) appropriate, b), c) and f) described in nucleic acid molecules, to generate amplicon；And
H) the blood cell amount of each volume sample is determined based on the analysis amplicon.
Preferably, nucleic acid is plasmid, such as the plasmid such as bacterial plasmid of linearisation, such as pUC.
In this aspect of method, the nucleic acid (plasmid) for the bisulfite conversion that use first is simulated through computer will be expanded
Standardization, the nucleic acid (fall all CpG dinucleotides comprising demethylation standard gene (such as GAPDH), " artificial sequence "
It is set to the sequence of GpC) and blood cell specific gene (" specific gene ").All 3 kinds of ingredients in the nucleic acid with
There is (equimolar) in equivalent, and the bisulfite through computer simulation converts.It therefore, can be by standardized curve and corresponding
Calibration curve and sample directly compared with, and can be true by the ratio of blood cell specific gene and demethylation standard gene
Determine Relative cell counts.However, nucleic acid and " true " sequence is not corresponded to, because each C is replaced by T.The series such as referred to is dilute
It releases and the measurement of every kind of concentration of all genes generates the calibration curve of measurement.
For the accuracy of improvement method, using the second nucleic acid (plasmid), it includes demethylation standard gene (such as
GAPDH), " artificial sequence " (all CpG dinucleotides are inverted to the sequence for GpC) and blood cell specific gene are (" special
Property gene ").However, these sequences are converted without the bisulfite of computer simulation, and correspond to genome sequence (model
Enclose to reach and match object with genome, see below), therefore can be only used for measurement amplification (such as qPCR) efficiency.
The reason of second reference substance is dual.A) quantitative in order to confirm, it needs identical with biological sample to be analyzed
Reference substance (this is also that regulation requires).However, sequence and the single-stranded sequence rich in U in the first nucleic acid, by double-strand rich in AT
Compare.Only " real " the bisulfite conversion of double-strandednucleic acid allows the confirmation to compare.Then, it is standardized using the first nucleic acid,
The quotient that the bisulfite of blood cell specific gene and demethylation standard gene converts, gives efficiency factor.For
The weight divided by demethylation standard gene is converted based on the bisulfite that all CpG dinucleotides are inverted to the sequence for GpC
The quotient of sulphite conversion is same.
Preferably, " artificial sequence " (all CpG dinucleotides are inverted to the sequence for GpC) is comprising C and CpG sequence
The random sequence being not present in human genome of (being converted for bisulfite).In one embodiment, artificial sequence
It is the precise sequence of the part of the GAPDH (amplicon) of amplification, wherein CpG sequence is squeezed as GpC sequence." artificial sequence " is deposited
It is all 3 kinds of nucleic acid as described above, i.e., in the first nucleic acid (the bisulfite conversion through computer simulation), the second core
Acid (being converted for bisulfite) and (it is the sequence uniquely analyzed) third nucleic acid (bisulfite simulated through computer
Conversion) in.
Third nucleic acid is given with determining amount into the blood for determining amount, then analyzed (such as purifying, weight sulfurous
Hydrochlorate processing, secondarily purified, desulfonation, specific amplification).Then, (how many copies are standardized to the first nucleic acid
It is measured and gives into reaction), using determining efficiency compared with the second nucleic acid, and (remaining) is determined using third nucleic acid
Copy number.The loss of the genomic DNA of any loss program identical as experience is compared.Overall process allows accurate confirmation
With DNA described in absolute quantitation, and pass through the cell in this such as blood sample of whole blood.
In one embodiment, the present invention relates to artificial sequences, for the essence of the part of the GAPDH (amplicon) of amplification
True sequence, wherein CpG sequence is squeezed as GpC sequence, to implement tool when method of the invention.
The component of cell immune system remains the valuable diagnostic message for various disease.It is thin for quantitative immunological
The reference substance technology of born of the same parents' monitoring is flow cytometry.However, this method is limited to the blood that wherein cell integrity is maintained
Sample.In routine clinical program, analysis is limited to fresh blood samples as analysis substrate by this reality.
In order to widen the use scope of diagnosis immunologic surveillance, inventor realizes epigenetic qPCR system for quantitatively leading
The leucocyte group wanted.After the methylation signature for determining immune cell type specificity, analyze from 25 healthy donors, 97
The whole blood of name HIV patient and neonatal 325 Guthrie cards are come from, including with 25 of primary immunodeficiency (PID)
Card.The flow cytometry and epigenetic of B cell, NK cell, total T cell, t helper cell and cytotoxic T cell has been determined
Methodology consistency between data, and challenged the ability of new technology identification quantitative immunological cell defect.
Data are shown, are tested according to Bland-Altman, are existed through epigenetic qPCR measurement and the quantitative of flow cytometry
Implement to be equivalent in health objects and HIV patient.Epigenetic is quantitatively white thin suitable for fresh and frozen blood sample
The opposite and absolute frequency of born of the same parents' subgroup.With flow cytometry on the contrary, the immunocyte analysis of Guthrie card accurately identifies new life
PID case in youngster.The epigenetic for implementing immunocyte group is quantitative equivalent with standard flow cytometry height, this is provided more
Large-scale possible application, including the dry blood cake of analysis may mention for patient from far-off regions or from the counting of neonatal blood
Approach is supplied.
Preferred method is according to the present invention, wherein the blood immune cells are thin selected from leucocyte, T- lymphocyte, grain
Born of the same parents, monocyte, B- lymphocyte and/or NK cell.
Preferred method is according to the present invention, wherein the recombinant nucleic acid molecules are selected from plasmid, yeast artificial chromosome
(YAC), human artificial chromosome (HAC), the artificial chromosome (PAC) in the source PI, bacterial artificial chromosome (BAC) and PCR are produced
Object.
Preferred method is according to the present invention, wherein the demethylation standard gene is selected from all cells to be detected
The gene of middle expression such as, such as house-keeping gene, e.g., such as GAPDH.
Preferred method is according to the present invention, wherein the blood cell specific gene is selected from all blood to be detected
The gene expressed in liquid cell such as, such as CD4.
Preferred method is according to the present invention, wherein the blood sample is selected from peripheral blood, capillary blood or venous blood
Sample or more than part such as, such as peripheral blood mononuclear cells, clot and dry blood cake.
Preferred method is the step for further including the immune state based on the quantitatively deduction mammal according to the present invention
Suddenly.
Another aspect of the present invention is related to diagnostic kit, and it includes the materials for implementing method of the invention, optionally
Together with operation instructions.Preferred material is nucleic acid molecules and/or bisulfite agent.
Another aspect of the present invention is related to kit of the invention and is implementing the application in method of the invention.
Current immunocyte monitoring needs are fresh or save good blood sample.It is used for here, inventors herein proposing
The substitute technology of difference blood cell counting allows to calculate blood fresh, that formalin is fixed, freezing or dry
Opposite and absolute cell numbers in sample.
Epigenetic qPCRs occurs as a kind of very promising tool, does not need complete cell or high standard
It saves.The data (14) of the demethylation of FOXP3TSDR show such epigenetic cytometer in report regulatory T cells
Several feasibilities.
In the present invention, it is resolved about still outstanding question below: a) several important cell classes
The availability of the possible epigenetic marker of type, b) mark between various cell type specificity epigenetic qPCR systems
Standardization, c) provide epigenetic signal confirmation quantitative (17) method, d) relative to given blood volume quantify and e) with
To detect the methodology consistency of the FCM goldstandard technology of corresponding immune cell type.
For cell type identification ideal DNA methylation marker target cell type (close to 0% methylation) and
It is distinguishing between every other cell type (close to 100% methylation).Here, having studied for immunocytochemistry
Marker applies the non-fully quantitative approach of bisulfite sequencing in the identification site of presumption for the first time.These data are shown
The site of the immune cell type of all tests temporarily meets the standard of ideal marker.Selection discrimination property CpG- dinucleotides is used
It is measured in qPCR and develops and methylation and demethylation template variant is tested.Realize the effective of target DNA and
Quantitative amplification, and do not have to background of the detection from non-target template.QPCR measures Robust Performance, in fresh or frozen blood
Deviation is low.
For testing a variety of different cell types in heterogeneous samples simultaneously, methylated using locus specificity
The qPCR of (i.e. CpG) and demethylation (TpG) is not optimal.The amplification efficiency of every kind of qPCR system is for example due to different
CpG density and it is different, therefore for the no biology of all measurements or technically stable parameter (20).On the contrary, by special base
Because the regulation and control fragment (regulatory stretch) of GAPDH (21) always demethylation is used as fixed denominator.In this way,
Quantity relative to demethylation site in GAPDH counts every kind of cell type-specific site, that is, assumes have all
In nucleus.However, its specificity epigenetic site at test purifying cell type show, use GAPDH as divide
What the amplification system of female quantitative deviation locus specificity methylation obtained quantifies.These deviations depend on individual sites and expansion
Increasing system (22,23) shows that be standardized can not be complete with the standard curve from the plasmid through computer simulation conversion
Offset the amplification difference of the actually DNA of bisulfite conversion.Plasmid DNA is double-strand, and includes beyond for apparent
The GC (24) of the template of genetic amplification about 40%.On the contrary, bisulfite conversion DNA be mainly it is single-stranded, after amplification
G/C content significantly reduces.In addition, the latter has been exposed to the harsh chemical treatment for leading to fragmentation, this can not be physically
Cytosines by the demethylation simulated through computer are thymidine to represent.
In order to compensate for the effect, by the genome sequence of the original demethylation containing GAPDH and all immunocyte classes
The single plasmid molecule (" caliberator ") of the marker gene of type specificity is handled together with biological sample.The caliberator provides
The opposite and equimolar of the DNA of bisulfite conversion by different qPCR systems is quantitative.However, it does not replace standard
Plasmid, because accurate independent quantitative is a challenge in bisulfite conversion process.Therefore, the weight through computer simulation is sub-
The plasmid (" reference substance ") of sulfate conversion continues on for copy number and determines.Caliberator plasmid provides the height in various experiments
Repeatable measurement, and compensate for the efficiency variance between the GAPDH observed and locus specificity measurement.In short, reference substance
Become epigenetic qPCR relative to all (demethylation) GAPDH copy respective with being used in parallel for caliberator plasmid
Site in demethylation confirmation quantitative (17) method.
It is in Fig. 1 and table 1 statistics indicate that, the analysis in differential methylation site is immune thin for what is detected and quantitatively determine
The strong substitution of born of the same parents' type.However, other than cell type specificity, if it is desired to carry out cell in this way
It counts, then needs to be determined by experiment the direct proportionate relationship of cell type and DNA copy number.Such direct linear correlation can
Can by blood platelet, granulophilocyte residual DNA or DNA copy and cell number between convert and influenced.Therefore, pass through table
The cell quantification and FCM analysis for seeing heredity qPCR carry out Orthogonal contrasts.When using the confirmation relative quantification introduced in this research,
Respective cells type and the data of marker are slightly different, and system deviation range is -6% to+11%.However, on the whole,
Two kinds of technologies observe higher methodology consistency between selected individual markers as a whole.
About routine clinical application, WHO approves that versus cell type is quantitative in HIV therapy guide, but medical reality is wanted
Ask the cell count (25,26) of each volume.For epigenetic immunocyte parting, which results in problems, because DNA is recycled
It is completely not quantitative, and the relationship between amount of DNA and blood volume is not determined biologically.However, falling for concentration will be determined
GAPDH variant (the GAP set[GC]) mark-on allow into blood sample approximate inference DNA extract before original DNA content.To the greatest extent
The different efficiency (27) of genomic DNA and Plasmid DNA have been described in pipe, but such difference in bisulfite processing and produces
It may be substantially reduced after raw fragmentation.In order to assess mark-on concept, by epigenetic immunocytochemistry and same blood is come from
The flow cytometry data of sample carries out parallel.About the deviation and the limit (28) of consistency, compared to previous publications based on anti-
Methods comparison (29) between the distinct methods of body, the data presented show the distribution of uniform error and it is smaller partially
Difference.Therefore, inventor's statistics indicate that, WBC sub-population can be reliably detected by epigenetic immunocytochemistry, and
And it is almost suitable with FCM data.
The inherent challenge of quantitative Diagnosis marker is its unfailing performance except normal physiologic range, and is protected from easy
The sample deposited.Quantitative for immunocyte, PID constitutes such situation.Patient suffers from serious immune deficiency, and passes through
The quantitative cell counting of FCM is infeasible to the dry blood card for neonatal screening.
In this case, the diagnosis of new method can be best informed compared with the diagnosis performance of goldstandard method
Ability.Because direct method compares since TREC is counted compared to by between FCM and/or the cell quantification of epigenetic qPCR
Different measurement parameters and it is infeasible, such result relatively seems indispensable.Epigenetic immunocytochemistry can be from dry
Blood cake reliably identifies all PID patients, including XLA patient.In addition, it provides related NK cellular level (potential gene
The important hints of defect, especially ADA-SCID) information because these delay mode of onset SCID in, NK cell (with
The SCID of other forms is opposite) missing.
In short, present invention demonstrates that, epigenetic cell count provides the accurate and accurate method for immunologic surveillance,
Either measured as the continuous parameter of percentage or absolute cell count measurement or as sorting parameter.It is lower when detecting
Method consistency when, as observed by between the epigenetic to monocyte and FCM marker, this shows given cell
Heterogeneous or indefinite marker specific effect consistency in group.
To sum up, the present invention is steady by the height for providing the blood analysis sample that can be saved from micro simplicity
Platform highlights the applicability that epigenetic immunologic surveillance is used for immunodiagnosis as valuable diagnostic method.
The present invention will be explained further in the following example and in the accompanying drawing now, but its is without being limited thereto.For of the invention
Purpose, during all references cited herein is incorporated herein by reference in their entirety.
Fig. 1 shows the DNA methylation spectrogram of the marker gene in the immunocyte group of purifying.Matrix shows 7 kinds
The cell type-specific DNA methylation mode of marker gene and reference gene GAPDH.In a matrix, immunocyte class
Type is to arrange arrangement, as shown in x-axis.Gene and corresponding amplicon (Amp) are shown at y-axis.Gene is separated by red line,
Every row represents the single site CpG.The CpG methylation level of measurement is according to range from yellow (0% methylation) to blue
The color scale of (100% methylation) is color coded.
Fig. 2 shows the schematic overview of the different basis weights method of epigenetic cell count.For all methods, invention
People assumes than 1 cell (2-allels-to-1-cell) relationship of 2 simplified allele.The base respectively analyzed in the present invention
Because of autosomal (i.e. diploid), and have shown a species specific cell type (in this scenario: CD4+T is auxiliary
The site CD4 in synergidae) in for demethylation, and in every other blood cell type be exhaustive methylation.
A the quantitative process of locus specificity relative percentage) is illustrated.The demethylation with unknown number (#) and methylation
Diploid gene group DNA copy blood sample in, by only by the Cytosines of demethylation at uracil, weight sulfurous
Epigenetic methylation state is converted into original sequence by hydrochlorate conversion.It is methylation and demethylation in the position CpG
Cytimidine occurs according to the gene regulation of specificity.The ura DNA of conversion is the CpG methyl by qPCR specific amplification
Change state, thus the uracil base-pair with adenylate is generated at the region of original demethylation comprising TpG dinucleotides
Amplified matter.Then, qPCR allows to pass through amplification based on the plasmid through computer simulation conversion being serially diluted is calculated
(f) linear interpolation (f-1) is to copy counting number.By the interpolation copy number of the copy of original demethylation at the site divided by
All copies at the site, that is, copy methylate and demethylation, to calculate the relative percentage at genomic locus
Methylation.Conversion in biological sample disturbs the integrality of genomic DNA, and plasmid represents amplified production rather than substrate.It is logical
Cross the difference that unknown " transforming factor (CF) " provides obtained amplification efficiency.It is relied on when comparing with seldom methylation state
It is considered insignificant when the amplification of two very high homology sequences of property SNPs.For general relative percentage qPCR
(B), the gene about the plasmid control object, its interpolation and cell type specificity demethylation that use through computer simulation conversion
Site, it is quantitative using the epigenetic of same principle.The site of cell-specific demethylation is not assessed, but expands generation
The site GAPDH of the general demethylation of all cells of table.Quantitative reference is used this as, the site of all specificity can mark
Standard turns to full gene group copy count.It cannot assume that CF is similar in this case, because between different sequences not
It is assumed that homology.Therefore, the opposite demethylation of every kind of all cells is by there are the interference of different CF.To offset different conversions
The influence of efficiency, by C) shown in introduce caliberator plasmid.Its contain equimolar all relevant cell specific positions and
The genome sequence of GAPDH.The interpolation of amplification, which provides, interferes the different copy numbers for converting specific efficiency.Different copy numbers
Ratio provides efficiency factor (EF), can be used for eliminating and converts relevant difference between reference substance and sample.EF is incorporated to (B)
In to provide confirmation copy number quantitative.D) for the counting of the cell of each volume blood, it is known to determine that the blood of amount is supplemented with
The plasmid of the dDNA sequence (GAP-GC) containing synthesis of copy number.Use the phase of the plasmid through computer simulation conversion
Quantitative and EF is calculated by operation as shown above.The GAP-GC for being inserted into initial amount allows monitoring of DNA to prepare, convert, and qPCR is mentioned
The good prediction object for the treatment of effeciency is supplied.Then the blood cell for calculating initial amount is possibly realized.
Fig. 3 show compared by flow cytometry and epigenetic qPCR it is immune thin from 25 healthy donors blood
Born of the same parents are quantitative.By the immunocyte such as through flow cytometry (y-axis) measurement to the epigenetic through immune cell type specificity
The analog value that qPCR analysis (x-axis) determines makees scatter plot.A relative immunity cell count) is shown, intermediate value is in total leukocyte
Percentage provide.Linear Pearson correlation coefficient is r=0.95.B absolute immunity cell count) is shown, intermediate value indicates
For the cell number of every μ l blood, it is characterised in that correlation r=0.95.Red line indicates the recurrence by all data point calculations
Line, black line indicate bisector.Symbol in right block indicates different individual immunity cell masses.
Fig. 4 shows the method ratio in HIV group between the flow cytometry of T cell subgroup and epigenetic qPCR analysis
Compared with.A the comparison (indicating with % relevant to total karyocyte) of relative immunity cell count) is shown.Big figure is shown through table
See the scatter plot of three kinds of T cell groups of heredity qPCR analysis (x-axis) and flow cytometry (y-axis) analysis.Black line and red line difference
Represent bisector and the tropic.Linearly dependent coefficient according to Pearson is (p < 0.0001) r=0.982.Small figure is shown
Bland-Altman analysis, wherein average cell meter that will be average between every kind of epigenetic and cell count measurement (x-axis)
Number (being indicated with %) maps to its (opposite) difference (y-axis).In every width Bland-Altman figure, the reflection of upper and lower part red line
The limit of consistency, and the red line at center shows system deviation.Above and below every red line, 95% confidence interval
Appear dimmed dotted line.Top Bland-Altman figure: total T cell；Deviation: 6.43%；Consistency lower limit: -9.15%；Unanimously
The property upper limit: 22.02%.Middle figure: cytotoxic T cell；Deviation: 11.23%；Consistency lower limit: -15.36%；In consistency
Limit: 37.83%.Bottom right: T helper cell；Deviation: -6.04%；Consistency lower limit: -41.34%；The consistency upper limit:
29.25%.B the comparison of absolute immunity cell count (such as indicating with the cell of every μ l blood)) is shown.Left side: scatter plot point
Analysis；Pearson r=0.955 (p < 0.0001).Right side: Bland-Altman analysis: upper figure: total T cell；Deviation :-
4.76%；Consistency lower limit: -39.62%；The consistency upper limit: 30.09%.Middle figure: cytotoxic T cell；Deviation: 0.03%；
Consistency lower limit: -35.78%；The consistency upper limit: 35.83%.Bottom right: T helper cell；Deviation: -17.61%；Under consistency
Limit: -59.68%；The consistency upper limit: 24.46%.
Fig. 5 shows that (and CD4 hereafter and CD8T is thin using CD3+T cell, CD19+B cell, CD56+NK cell
Born of the same parents) epigenetic analysis of markers control Guthrie card and PID it is sick.Normal healthy controls are provided with ash point, and are converted from log
Copy number estimated bivariate normal distribution 99% (blue) and 99.9% (red) confidence interval.Test the measurement of group
Data are provided as number, and are color coded according to phenotype.
Embodiment
It writes a Chinese character in simplified form: Amp, amplicon；QPCR, quantitative real-time polymerase chain reaction；FCM, flow cytometry；HSCT, hematopoiesis
Stem cell transplantation.RDls, the opposite demethylation of locus specificity；RDu, general opposite demethylation；DDu, general confirmation
Demethylation；LC, white blood cell count(WBC)；CF, transforming factor.
Leucocyte group.Peripheral blood blood sample is obtained from healthy donors, and living by the high speed fluorescence of (16) as discussed previously
Change cell sorting methods and is divided into CD15+Granulocyte, CD14+Monocyte, CD56+Natural killer cells, CD19+B- lymphocyte,
CD3+CD4+T- auxiliary cell and CD3+CD8+Cytotoxic T cell.Purity by the sorting cell of Flow Cytometry Assay is
> 97%, and vigor is > 99%.
Peripheral blood whole blood sample.The anticoagulant peripheral blood sample acquisition of EDTA- (comes from each blood from 25 health objects
Liquid extract), German outpatient clinic receive treatment 97 HIV+Patient (each a blood extract) and 26 are from Sheng Lafei
(acute myelogenous) for receiving hematopoietic stem cell transplantation of your university hospital (San Raffaele University Hospital)
Leukaemic.From latter group, 92 blood have been carried out from 180 days after the conditioning stage to transplanting and have been extracted.To all samples
It is parallel to carry out epigenetic qPCR analysis and standard flow cytometry analysis, with quantitative (seeing below) for immunocyte, and nothing
Additional venipuncture need to be carried out according to medical device act (Medical device act).It is same that associated mechanisms give ethics
Meaning.Epigenetic is analyzed, all data all set experimenter blind.For diagnostic facs analysis, sample does not set blind.
DNA preparation.Sequencing and qPCR analysis for the immunocyte of purifying, use DNeasy Tissue kit
(Qiagen) genomic DNA is separated according to manufacturers instruction.In every other application, single tube dissolution is carried out to blood sample
It is converted with bisulfite, without DNA preparation before.
Bisulfite conversion.In order to convert the genome or Plasmid DNA of purifying, EpiTect is used according to manufacturers protocol
Fast bisulfite conversion reagent box (Qiagen).It is in order to which direct bisulfite converts whole blood, the EDTA of 20 μ l is anticoagulant
Blood blood (or caliberator plasmid) and 16 μ l lysis buffers, 3 μ l Proteinase Ks (Qiagen), and it is suitable when 1 μ l GAP[GC]
The mixing of substance grain is demarcated, 20,000 copy/μ l blood is generated, is then incubated for 10 minutes at 59 DEG C.For conversion, 90 μ l are added
Ammonium bisulfite 56to 70 (68-72%, pH 4.8-5.3, Chemos AG) and 30 μ l tetrahydrofurfuryl alcohols (Sigma-Aldrich).According to
The purifying for the DNA that " the quick bisulfite conversion reagent box of EpiTect " scheme is converted and converted.
Bisulfite sequencing.Including 1x PCR buffer, 1U Taq archaeal dna polymerase (Qiagen), 200 μM
25 μ l final volumes of the genomic DNA of the bisulfite of each forward and reverse primer of dNTPs, 12.5pmol and about 10ng conversion
Middle carry out PCR amplification, response procedures be 95 DEG C of 15 minutes and 40 recycle 95 DEG C 1 minute, 55 DEG C 45 seconds and 72 DEG C 1 point
Clock and finally extension step 72 DEG C 10 minutes.Using ExoSAP-IT (USB Corp.) purified pcr product, and use a kind of PCR
Primer is sequenced using ABI Big Dye Terminator v1.1-chemistry (Applied Biosystems), then exists
Capillary electrophoresis is carried out on 3100 Genetic Analyser of ABI.AB1 file is interpreted using ESME (18).
Epigenetic qPCR analysis.Using Roche LightCycler 480Probes Master chemistry 10
It is tested in the final volume of μ l, it includes 50ng lambda phage DNA (New England Biolabs) and up to 100ng to turn
The DNA profiling of change or enough plasmids.The normal concentration of each primer is 1.5 μM, (0.75 μ other than genome demarcates substance grain
M)。CD4+T cell TpG measures (4.5 μM of forward primers；3 μM of reverse primers).Standard probe concentration is 0.25 μM, in addition to CD4+T
Cell, CD8+T cell, NK cell and calibration substance grain are outer (for the system of TpG- specificity, each 0.125 μM of probe).It is hot general
Condition be 95 DEG C 10 minutes, then 50 circulation 95 DEG C 15 seconds and 61 DEG C 1 minute.
Plasmid.The sequence that two kinds of bisulfites corresponding to methylation or demethylation marked region convert is through counting
The board design of calculation machine is synthesized and is inserted into plasmid pUC57 (Genscript Inc.), and is used as the positive control that measurement is established
With the quantitation standard of qPCR experiment.Standard plasmid has all measurement target sequences (as TpG or CpG variant), and through dividing
Connection, provides equimolar all measurement targets in sub.Plasmid is linearized through spectrophotometric standard measure by Sca I, and
Serial dilution in the lambda phage DNA (New England Biolabs) of 10ng/ μ l, with obtain it is each reaction 31250,6250,
1250, the quantitation standard of 250,50 or 30 copies.QPCR is standardized, single caliberator plasmid, equimolar are generated
Ground includes all measurement target sequences of the unconverted demethylation form of genome.It is quantitative for the leucocyte of every μ l blood,
Calibration (spike-in) plasmid for carrying unconverted artificial GAPDH gene region is designed and produced, is corresponding exactly to
The target of GAPDH specificity qPCR measurement, but it is GpCs (GAP that all CpG dinucleotides, which are inverted,[GC])。
Oligonucleotides.Positive (fp), reversed (rp) primer and hydrolysis probes (p) (Metabion AG) by its relative to
Human genome assembles the chromosome location of GRCh38.p5, Release 84 (March 2016) to indicate.For weight sulfurous acid
The oligonucleotides of salt sequencing: AMP1255:fp:12:6790192-214, rp:12:6790582-603；AMP1730:fp:9:
128149251-72,rp:9:128149589-609；AMP2000:fp:12:6790724-46,rp:12:6791160-80；
AMP2001:fp:12:6791141-62,rp 12:6791535-60；AMP2007:fp:2:86821232-54,rp 2:
86821674-95；AMP2178:fp:6:161375641-62,rp6:161376086-108；AMP2249:fp:11:
68371460-81,rp:11:68371926-47；AMP2674:fp:16:88653882-902,rp:16:88654299-
88654320.Oligonucleotides for qPCR analysis: CD4:TpG:fp:12:6790871-98, rp:12:6791046-73, p:
12:6790998-1019；CpG:fp:12:6790871-900,rp:12:6791046-72,p:12:6790997-1020.
CD8B:TpG:fp:2:86821374-1400,rp:2:86821476-93,p:2:86821425-52；CpG:fp:2:
86821372-1401,rp:2:86821463-83,p:2:86821425-55。LPR5:TpG:fp:11:68371608-28,rp:
11:68371721-45,p:11:68371666-84；CpG:fp:11:68371611-35,rp:11:68371720-48,p:11:
68371662-86.MVD:TpG:fp:16:88654112-36,rp:16:88654173-90,p:16:88654136-55；CpG:
fp:16:88654111-36,rp:16:88654172-89,p:16:88654136-58。PARK2:TpG:fp:6:
161375730-55,rp:6:161375851-66,p:6:161375804-25；CpG:fp:6:161375784-807,rp:6:
161375851-70,p:6:161375805-830。LCN2:TpG:fp:9:128149258-78,rp:9:128149353-75,
p:9:128149289-309；CpG:fp:9:128149257-77,rp:9:128149353-76,p:9:128149287-309.
CD3+T cell and GAPDH specific amplicon and the oligonucleotides of qPCR system had previously delivered (15).
The flow cytometry characterization-of whole blood sample in order to more apparent genetic analysis and standard flow cytometry as a result,
It is measured after lysed erythrocyte by MACSQuant hemacytometer (MiltenyBiotec, Bergisch Gladbach)
CD45+The absolute quantity of leucocyte.In addition, calculating CD15 by previously described (14,32)+Granulocyte, CD14+Monocyte,
CD19+B cell, CD56+NK cell, total CD3+T cell and CD4+And CD8+The frequency and absolute counting of subgroup.
Statistical analysis-calculates aggregation duplicate measurements three times by the maximum second derivative method using LC480 software (Roche)
CP (" crosspoint "), with from using the reference substance based on plasmid dilution generate calibration curve by amplification (f) insert
Enter (f-1) and generates copy number (" plasmid unit ").Method between flow cytometry and measuring technique based on qPCR compare by
Following to carry out: the bivariate data from two methods are drawn in scatter plot.Linear regression is carried out to test: a) being different from 1
Slope and b) be different from 0 intercept.Check Bland-Altman map analysis deviation and accuracy statistics (28).Acceptable essence
Exactness is regarded as a departure from the average value percentage of deviation, reflects the inside limitation to the coefficient of variation of intraassay performance,
I.e. 0.2.This is converted into the acceptable 0.4 consistency limit.Inventor has reported the deviation of estimation, accuracy counts and respective
95% confidence interval.For correlation, Pearson product moment correlation coefficient has been used.It uses Cohens-Kappa coefficient (19)
Assess Rater consistency.All p values are all bilaterals.Using statistic software R 3.3.0.
Cell type-specific bisulfite conversion.CpG- dinucleotides is analyzed by bisulfite sequencing (18)
The conversion of methylation dependence, it is therefore intended that the immunocyte group that is sorted from human peripheral of identification.Candidate locus is selected from document
Or the discovery experiment from genome range.As CD4+The possibility marker of t helper cell, inventor devise three kinds of amplifications
Sub (Amp) is analyzed for bisulfite sequence, covers the controlling element in the 5 ' region of First Intron of CD4 gene
(Amps1255,2000 and 2001).It is converted in bisulfite and only in target cell, that is, CD3+CD4+After being expanded in T lymphocyte,
The unmethylated site CpG is detected as TpG residue.Identical CpG is to bisulfite conversion in lazy in control cell type
Property, including CD56+Natural kill (NK) cell, CD3+CD8+T lymphocyte, CD14+Monocyte, CD19+Bone-marrow-derived lymphocyte and
CD15+Granulocyte (Fig. 1).Amplicon (the Amp that inventor passes through the design Targeted-control element in its third introne
2007) CD8B gene, is had studied as CD8+The potential apt epigenetic of cytotoxic T cell marks.Here, weight sulfurous acid
The CpGs that salt mediates is converted only in CD3+T CD8+It is observed in (target) cell, and CpGs is in conversion in control cell type
Inertia.Similar to CD8+And CD4+The data of T cell, inventor identify epigenetic label, and each epigenetic label is in target
It is uniquely demethylation in cell type, and is exhaustive methylation in control leucocyte group accordingly.Correspond to
The amplicon of gene LRP5 (Amp2249) and MVD (Amp2674) are used separately as the epigenetic marker of B cell and NK cell.
Prior disclosure composition CD3+Between the gene of the marker of T cell the region CD3G and CD3D (Amps 1405,1406 and 1408)
The methylation spectrogram (15) of DNA methylation spectrogram and GAPDH (Amp 1570).
The opposite qPCR of locus specificity is measured.The position CpG for targeting above-mentioned otherness methylation, such as institute in method part
State design quantitative PCR measurement system.QPCR system is characterized in that being cloned into the heavy sulfurous acid simulated through computer in plasmid
The template DNA (Fig. 2A, right figure) of salt conversion.It is general for TpG template (CpGs of the demethylation in simulation genomic DNA)
Plasmid carry equimolar stoichiometry targeting regions for all measurements and artificial GAP[GC]Sequence (general TpG plasmid),
And it is each amplicon individually designed " CpG plasmid " (CpG of the methylation in simulation genomic DNA).In mutually contradictory mould
Observe that high-tech specificity does not have cross reactivity (table 1, " control based on plasmid ") in plate.By the way that corresponding expansion will be come from
The PCR signal for increasing (f ') is associated with the amplification (f) of the plasmid of serial dilution, to estimate the original of the gene order in blood sample
Beginning copy number (Fig. 2A).Biological characteristis specificity is tested for the immunocyte group of purifying.For target cell type, TpG
With high and low copy number is observed in CpG system respectively.On the contrary, having found low copy in TpG system for control cell type
Shellfish number, and high copy number is had found in CpG system.Respective gene loci (RDls) at opposite demethylation it is thin in target
Range is 89.9% to 100% in born of the same parents' type, and in control is 0.0% to 3% (table 1).For the CD4 of purifying+T cell is seen
Exception is observed, 8.9% demethylation, and vice versa (i.e. CD8 are shown at the site CD8B+9.6% CD4 is de- in T cell
Methylation).
General and confirmation is quantitative.It is in order to provide the quantitative commbined foundations of all cells, the GAPDH of demethylation is special
Property amplification together with the TpG system of above-mentioned cell-specific analyze (Fig. 2 B).General TpG plasmid is as amplification reference substance.It utilizes
This mode, by determining the sample amplification f ' of every kind of marker and GAPDH and reference substance amplification (f) is associated in sample
General opposite demethylation (RDu).Inventor's statistics indicate that, RDuIt is not always and corresponding locus specificity demethylation
(RDls) matching.It in order to compensate for the intrinsic system change, uses " caliberator plasmid ", not with equimolar amounts and for it
All measurement targets of conversion (i.e. demethylation) state.Estimated based on unconverted plasmid in the mark based on reference substance plasmid
The efficiency variance between single qPCR system retained after standardization, and generate qPCR efficiency factor (EF).In about 25 experiments
Determine it is each it is cell type-specific measurement GAPDH between average EF, and range be for CD4 0.53 to for
1.17 (the table 1.EF) of CD3x and y.Then, EF is in general opposite demethylation (RDu) on application allow general confirmation piptonychia
Base quantifies (DDu；Fig. 2 c).
There is the artificial GAPDH sequence (GAP all CpG dinucleotides being inverted as GpC by introducing[GC]) " calibration
Plasmid " and corresponding qPCR measurement are to establish absolute quantitation.GAP[GC]The substrate specificity of the qPCR measurement of specificity is coming from
Contain and without calibration substance grain whole blood bisulfite conversion DNA on confirmed, wherein be not detected with it is interior
The cross reactivity of source property GAPDH gene.On the contrary, when containing GAP[GC]The calibration plasmid template test GAPDH of sequence is special
Property qPCR measurement when, amplified signal is not detected, this also demonstrates high substrate specificity, this is for absolute quantitation must can not
Few.For immunocytochemistry, calibration plasmid is added in blood sample, generates and each gives initial sample volume really
Determine concentration (Fig. 2 D).In addition, including on the plasmid control object and caliberator plasmid of the bisulfite conversion through computer simulation
Artificial GAP[GC]Sequence, obtaining the respective equimolar factor (EF) for correction is 0.87 (GAP[GC]；0.83/0.92).
Pass through the comparative immunocytochemistry of flow cytometry and epigenetic qPCR.It supplies from 25 adult healthies
The blood sample of body carries out standard flow cytometry (FCM) and epigenetic qPCR, to be used for CD15+Neutrophil leucocyte, CD14+
Monocyte, CD19+B cell, CD56+NK cell, CD3+、CD4+And CD8+T cell it is universal dosed.It will be from two methods
Data as comparative counting (Fig. 3 A) or absolutely (Fig. 3 B) to mapping each other.It is high-caliber consistent between two methods of scatter plot instruction
Property, relative quantification and absolute quantitation its Pearson correlation coefficient r for leucocyte group are 0.95 (p < 0.0001).Compare FCM
Bisector is not deviated considerably from the regression curve of epigenetic qPCR relative quantification.However, observing difference for absolute quantitation
Different smaller but dramatically different slope indicates proportional system deviation.
In order to test the new method of inventor in true clinical environment, inventor is measured from 97 HIV+Object
Blood sample, about CD3+、CD4+And CD8+Cell count counts to quantify by standard FCM and epigenetic.In the present invention
In, it is 0.91-0.98 (p < 0.0001) that correlation analysis, which obtains opposite and absolute quantitation Pearson r related coefficient range,
(Fig. 4).Absolute quantitation is based on GAP[GC]Plasmid mark-on is into blood sample, to apply GAP[GC]The qPCR of specificity measures system
System determines whole white blood cell count(WBC)s.The leukocyte count such as determined by FCM and epigenetic qPCR method is highly relevant
(Pearson r=0.8；p<0.0001).For appraisal procedure comparativity, inventor has carried out Bland-Altman analysis (17；
Fig. 4).For all 3 kinds of cell types, the systematical difference (deviation) between two methods is less than 11% (opposite) and 18%
(absolute).In addition, for all 3 kinds of markers, nonsystematic fluctuation is kept below when comparing FACS and epigenetic qPCR
25%, show that two methods inaccuracy level is low.According to these data, for all system types, FCM and epigenetic meter
Several biological readouts are seemingly good associated.Sample, which acquires, and pretreatment is not always ensures the blood of known quantity, such as
It does not include flow cytometry in dry blood cake.In these cases, in order to test the diagnosis accuracy of epigenetic qPCR,
Inventor determines acquisition from the Guthrie card of 250 healthy newborns and from suffering from SCID (x patient), ADA-SCID (y)
With the immunocytochemistry in 30 blood cards of the primary immunodeficiency disease patient of XLA (z).After an analysis, data are not
If blind, result is compared with the data obtained using TREC and KREC analysis and disclosed available genetic analysis.Such as
Shown in Fig. 5, in CD3 in GAPDH figure, there is 13 to show 99.9% confidence area of normal population in 15 SCID cases
Between, provide only positive diagnosis.O.11 case is located at except 99%CI in B cell analysis, and the position in NK cell analysis
Except 99.9%CI.No. 10 SCID is located within " normal population " of T cell analysis, but in B cell and NK cell pair
It is located at except 99.9%CI in GAPDH analysis.These combinations in No. 10 and o.11 SCID case clearly indicate that immune thin
The violent change of born of the same parents' steady state, and need complete screening post analysis.When the SCID case of analysis delay breaking-out, No. 23 position
Except 99.9% section, No. 30 is located at except 99%CI, and No. 28 case is seemingly unsuspicious in T cell analysis
's.However, all 3 cases detect except 99.9%CI in B cell analysis, and at least in NK cell count
Except 99%CI.Have been reported by the T cell in XLA patient and has been positioned horizontally in except 99%CI for No. 1 and No. 8 case,
But all 5 cases are located at except 99%CI in B- cell, wherein No. 1, No. 2, No. 6 and No. 8 is located at 99.9%
Except CI.Equally, NK cytology cases 8 and 15 are located at except 99%CI.In B cell analysis, No. 15 case is located at
Except 99%CI.Normal healthy controls (No. 12, No. 14 and No. 29) of the mark-on into invention test are located in all measurements
Within 99%CI, and for T cell from the previously-accepting patient's's (No. 13, No. 18, No. 20) for crossing stem cell transplantation
Control sample is located within 99%CI, but No. 18 is still accredited as " improper ".Not identifying in this analysis has obviously
Parent implantation SCID case because in the marker of all analyses its seem to be completely it is unsuspicious.Finally, CD4 and
The discovery of CD3 marker is supported in the analysis of cd8 cell part, but does not show significant single added value compared to CD3 screening.
The association analysis of GAPDH, CD3, B cell and NK raji cell assay Raji seem to provide the information of Precise Diagnosis.With will be in 3 series
Each of adjust to 99% or (99.9%) CI, inventor needs to correct multiplicity, and obtains 3% through Bonferroni correction
(0.3%) control of horizontal family error rate (family-wise-error-rate) (generalization of I class mistake).
Table 1
Table 1.RDls: the opposite demethylation (locus specificity) indicated with %；RDu: the opposite demethylation indicated with %
Change (general)；EF: efficiency factor；DDu: the confirmation demethylation (general) indicated with %
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Claims (11)
1. the quantitative methylation assay method of blood immune cells comprising following steps:
A) the human blood sample of the amount of determination is provided, it includes the diploid gene group DNA to quantitative blood immune cells；
B) recombinant nucleic acid for the bisulfite conversion simulated through computer is provided, it includes at least one demethylation standard bases
The sequence (" reference substance I ") that it is GpC that all CpG dinucleotides of cause at least one demethylation standard gene with described in, which are inverted,；
C) recombinant nucleic acid is provided, it includes at least one demethylation standard gene described in b) and b) described at least one is de-
All CpG dinucleotides of methylation standard gene are inverted the genome sequence (" caliberator of the demethylation for the sequence for being GpC
I")；
D) recombinant nucleic acid is provided, it includes all CpG dinucleotides of at least one demethylation standard gene described in b) to fall
It is set to the sequence (" calibration object I ") of GpC；
E) d of amount is determined to the sample addition described in a)) described in recombinant nucleic acid (" mark-on ")；
F) using described in bisulfite processing a) to the diploid gene group DNA of quantitative cell and c) and d) described in
Recombinant nucleic acid, by unmethylated Cytosines at uracil；
G) using primer pair amplifies a) appropriate, b), c) and f) described in nucleic acid molecules, to generate amplicon；And
H) blood immune cells (BIC) of every volume sample are determined based on the analysis amplicon.
2. the quantitative methylation assay method of blood cell comprising following steps:
A) human blood sample is provided, it includes the diploid gene group DNA to quantitative blood cell；
B) recombinant nucleic acid for the bisulfite conversion simulated through computer is provided, it includes at least one demethylation standard bases
Cause and at least one blood cell specific gene (" reference substance II ")；
C) recombinant nucleic acid is provided, it includes at least one demethylation standard gene described in b) and b) described at least one blood
The genome sequence (" caliberator II ") of the demethylation of liquid cell-specific genes；
D) using bisulfite processing a) described in quantitative cell diploid gene group DNA and c) described in recombinant nuclear
Acid, using by unmethylated Cytosines as uracil；
E) using primer pair amplifies a) appropriate, b), c) and d) described in nucleic acid molecules, to generate amplicon；And
F) the demethylation percentage (DDC) of every kind of all cells is determined based on the analysis amplicon.
3. the method for determining the absolute copy number of the methylated genes of immune cell type comprising:
A) implement method described in claim 1,
B) implement method as claimed in claim 2, and
C) identified BIC is multiplied with identified DDC.
4. method as claimed in any one of claims 1-3, wherein the blood immune cells are selected from: leucocyte, T- lymph
Cell, granulocyte, monocyte, B- lymphocyte and/or NK cell.
5. such as method of any of claims 1-4, wherein the recombinant nucleic acid molecules are selected from: plasmid, Yeast Artificial
Chromosome (YAC), human artificial chromosome (HAC), the artificial chromosome (PAC) in the source PI, bacterial artificial chromosome (BAC) and
PCR product.
6. method according to any one of claims 1 to 5, wherein the demethylation standard gene is selected to be detected
The gene expressed in all cells such as, such as house-keeping gene, e.g., such as GAPDH.
7. such as method of any of claims 1-6, wherein the blood cell specific gene is selected to be detected
Whole blood cell in the gene expressed such as, such as CD4.
8. such as method of any of claims 1-7, wherein the blood sample is selected from: peripheral blood, capillary blood
Venous blood sample or more than part such as, such as peripheral blood mononuclear cells, clot and dry blood cake.
9. the method as described in any one of claim 3-8 further includes quantitatively inferring the immune of mammal based on described
The step of state.
10. diagnostic kit, it includes the materials for implementing any one of claim 1-9 the method, optionally, and
Operation instructions.
11. the purposes that kit described in any one of claim 10 is used to implement the method for any of claims 1-3.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP17155496.7A EP3360971A1 (en) | 2017-02-09 | 2017-02-09 | Improved method for epigenetic immune cell counting |
| EP17155496.7 | 2017-02-09 | ||
| PCT/EP2018/053206 WO2018146209A1 (en) | 2017-02-09 | 2018-02-08 | Improved method for epigenetic immune cell counting |
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| CN110382713A true CN110382713A (en) | 2019-10-25 |
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| US (1) | US11319581B2 (en) |
| EP (2) | EP3360971A1 (en) |
| JP (1) | JP2020505938A (en) |
| CN (1) | CN110382713A (en) |
| AU (1) | AU2018217352A1 (en) |
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| DE102018116353B4 (en) * | 2018-07-05 | 2020-06-10 | Epiontis Gmbh | Method for the detection and enumeration of epigenetic immune cells in human blood samples for immunodiagnostics and newborn screening |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2199411A1 (en) * | 2008-12-16 | 2010-06-23 | Epiontis GmbH | Epigenetic markers for the identification of CD3 positive T-lymphocytes |
| CN103031372A (en) * | 2012-08-10 | 2013-04-10 | 深圳先进技术研究院 | ZNF545 gene methylation quantitative detection method |
| CN104046686A (en) * | 2005-04-15 | 2014-09-17 | Epi基因组股份公司 | Methods and nucleic acids for analyses of cellular proliferative disorders |
| WO2014170497A2 (en) * | 2013-04-19 | 2014-10-23 | Epiontis Gmbh | Method for identifying the quantitative cellular composition in a biological sample |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| DK2788510T3 (en) * | 2012-03-12 | 2017-03-20 | Epiontis Gmbh | Epigenetic marker for identification of CD3CD4-positive T lymphocytes |
| GB201221133D0 (en) * | 2012-11-23 | 2013-01-09 | Epiontis Gmbh | Epigenetic method for the identification of subpopulations of CD8 and T lympocytes, in particular CD8 alpha and beta T lymphocytes |
-
2017
- 2017-02-09 EP EP17155496.7A patent/EP3360971A1/en active Pending
-
2018
- 2018-02-08 US US16/484,246 patent/US11319581B2/en active Active
- 2018-02-08 EP EP18702740.4A patent/EP3580358A1/en active Pending
- 2018-02-08 JP JP2019542675A patent/JP2020505938A/en active Pending
- 2018-02-08 AU AU2018217352A patent/AU2018217352A1/en not_active Abandoned
- 2018-02-08 WO PCT/EP2018/053206 patent/WO2018146209A1/en unknown
- 2018-02-08 CA CA3051314A patent/CA3051314A1/en active Pending
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Patent Citations (4)
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|---|---|---|---|---|
| CN104046686A (en) * | 2005-04-15 | 2014-09-17 | Epi基因组股份公司 | Methods and nucleic acids for analyses of cellular proliferative disorders |
| EP2199411A1 (en) * | 2008-12-16 | 2010-06-23 | Epiontis GmbH | Epigenetic markers for the identification of CD3 positive T-lymphocytes |
| CN103031372A (en) * | 2012-08-10 | 2013-04-10 | 深圳先进技术研究院 | ZNF545 gene methylation quantitative detection method |
| WO2014170497A2 (en) * | 2013-04-19 | 2014-10-23 | Epiontis Gmbh | Method for identifying the quantitative cellular composition in a biological sample |
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| JALID SEHOULI等: "Epigenetic quantification of tumor-infiltrating T-lymphocytes", 《EPIGENETICS》 * |
| 李一君: "自身免疫糖尿病外周血CD4+T细胞DNA甲基化异常的研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
Also Published As
| Publication number | Publication date |
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| AU2018217352A1 (en) | 2019-08-08 |
| EP3580358A1 (en) | 2019-12-18 |
| EP3360971A1 (en) | 2018-08-15 |
| US20190390263A1 (en) | 2019-12-26 |
| US11319581B2 (en) | 2022-05-03 |
| JP2020505938A (en) | 2020-02-27 |
| WO2018146209A1 (en) | 2018-08-16 |
| CA3051314A1 (en) | 2018-08-16 |
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